# Haynes:Assembly101

(Difference between revisions)
 Revision as of 21:54, 20 January 2012 (view source)← Previous diff Revision as of 22:12, 20 January 2012 (view source)Next diff → Line 68: Line 68: # Calculate how many ng of insert you need to get a 2:1 ratio of insert molecules to 50 ng vector molecules
''X ng insert = (bp insert / bp vector) x 2 x 50 ng vector'' # Calculate how many ng of insert you need to get a 2:1 ratio of insert molecules to 50 ng vector molecules
''X ng insert = (bp insert / bp vector) x 2 x 50 ng vector'' # Calculate how many μL of insert and vector you will need for each ligation:
''X μL insert =  desired ng insert ÷ insert concentration ng/μL'' (do the same for vector) # Calculate how many μL of insert and vector you will need for each ligation:
''X μL insert =  desired ng insert ÷ insert concentration ng/μL'' (do the same for vector) - # Set up your digest reaction(s) as shown below: + # Set up your ligation reaction(s) in sterile 0.5 mL tubes as shown below: {| class="wikitable" width=350px {| class="wikitable" width=350px - | Plasmid DNA || 15.0 μl* + |   || Ligation || Negative Control || + | Insert DNA (X ng) || ___ μL || '''none''' |- |- - | Fermentas FastDigest enzyme 1 || 1.0 μl + | Vector DNA (50 ng) || ___ μL || same |- |- - | Fermentas FastDigest enzyme 2 || 1.0 μl + | 2x Roche Rapid Ligation buffer || 5.0 μl || same |- |- - | 10x FastDigest buffer + green loading dye || 3.0 μl + | NEB T4 ligase || 1.0 μl || same |- |- - | dH2O || 10.0 μl + | dH2O || ___ μL || ___ μL + insert volume |- |- - |   || 30.0 μl total + |   || 10.0 μL total |- |- - | colspan="2" | *For low yield DNA, use up to 25 μL and decrease dH2O accordingly.
Mix the reaction(s) thoroughly by flicking the tube.
Incubate at 37°C for 10 minutes. + | colspan="2" | Mix the reaction(s) thoroughly by flicking the tube.
Incubate at room temperature for 10 minutes. |} |}

+ '''Transform bacteria with the ligated plasmids''' ''30 minutes'' + # Warm selection agar plates at 37°C. + # Incubate DH5α Turbo competent cells on ice just until thawed. Use 30 μL per ligation. + # Add 30 μL thawed cells to the ligation reaction. Immediately place on ice and incubate for 10 min. (Do not heat shock; No 30 min. recovery is required for Amp resistance) + # Label the pre-warmed plates with the antibiotic name, strain name, ligation (e.g., "BB part A insert + BB part B vector"), your initials, and the date. + # Pipette the total volume of cells + ligation onto the agar; speared using sterile glass beads. + # Incubate overnight at 37°C to get colonies + Note: The negative control will show you the number of “background” colonies so that you can determine whether your transformation worked, or is just the result of vector self-ligation or selection failure.

## Revision as of 22:12, 20 January 2012

Model Procedure for Assembling Parts: Classic Ligation for Beginners
or, Cloning Sensei's Guide For the Aspiring Cloning Ninja

by Karmella Haynes, 2012

When I was a postdoc in Pam Silver's lab at Harvard (2008 - 2011), my lab mates and I generated large numbers of BioBrick assemblies so rapidly, and perhaps stealthily, that one of our colleagues in the department referred to us as "cloning ninjas." This guide is based on the MIT Registry of Standard Biological Parts suggested approach, which I've modified to make ligation-based assembly as quick and painless as possible. Let's begin.

Day 1*: Pick and amplify the desired plasmid DNA by growing transformed DH5α Turbo bacteria.

Make streaks from glycerol stocks 6 hours

1. Warm an agar plate at 37°C for at least 20 min.
2. Label the plate with the bacterial strain name (e.g., DH5α), the antibiotic, the BioBrick part(s) name, your initials, and the date.
3. Locate the desired -80°C glycerol stock. Use a sterile wooden toothpick to scrape up a tiny bit of the frozen bacteria and streak the plate.
4. Incubate the plate at 37°C for 6 hours to grow the bacteria.

Grow liquid cultures

1. Label 15 ml sterile culture tube(s) appropriately. Fill each tube with 2 ml of LB growth medium + appropriate antibiotic (e.g., 100 μg/ml ampicillin).
2. Using a sterile pipette tip, touch the bacterial streak (or pick up a single colony) and put the tip into the LB medium (bacterial end down).
3. Grow the cultures overnight in a shaking 37°C incubator.

*This may take two days instead of one if you're starting with a slow-growing strain.

Day 2: Extract the plasmids. Digest (cut), purify, and ligate (paste) the BioBricks. Put the assembled plasmid into bacteria

Extract the plasmid DNA: Qiagen Miniprep Kit 1.5 hours
To extract the plasmid DNA from the bacteria, perform a mini prep (refer to the Qiagen miniprep protocol). 2 ml of culture usually gives a yield of about 200 ng/μl (elution vol. = 75 μl).

Digest (cut) the DNA with restriction enzymes 30 minutes

1. First, write out a brief assembly strategy:
New Construct Name: BioBrick Insert Name, size (bp), cut sites + BioBrick Vector Name, size+backbone (bp), cut sites
2. Set up your digest reaction(s) as shown below:
 Plasmid DNA 15.0 μl* Fermentas FastDigest enzyme 1 1.0 μl Fermentas FastDigest enzyme 2 1.0 μl 10x FastDigest buffer + green loading dye 3.0 μl dH2O 10.0 μl 30.0 μl total *For low yield DNA, use up to 25 μL and decrease dH2O accordingly.Mix the reaction(s) thoroughly by flicking the tube.Incubate at 37°C for 10 minutes.

Separate the fragments via gel electrophoresis and purify the fragments 2 hours

1. Make a 0.8% gel: add 0.48 g agarose to ~60 ml 1x TAE buffer in a glass flask.
2. Mix by swirling and microwave for 40 seconds. Mix by swirling again (to eliminate air pockets and prevent boiling-over) and microwave for 40 seconds.
3. Set up a gel mold and comb. Make sure the teeth are the right size to hold 30 μL of sample.
4. Add 5 μl of 10 mg/ml ethidium bromide (etBr) to the agarose for a final concentration of ~0.8 μg/mL etBr. Mix by swirling (avoid making bubbles).
5. Pour the gel into the gel mold. Allow it to cool until it becomes opaque.
6. Fill a gel electrophoresis chamber with 1x TAE.
7. Remove the comb from the gel and carefully submerge the gel into the filled electrophoresis chamber.
8. Carefully pipette 15 μL pre-made 1 kb ladder mix into the first empty well and the DNA samples into the other empty wells.
9. Connect the electrical leads so that the positive end is at the bottom (DNA migrates to the positive end). Run the gel at 100 V.
10. Stop the gel when the yellow dye (Orange G) reaches the desired place on the gel (~1 hr.).
11. Remove the gel from the chamber and photograph under UV light.
12. Use a scalpel to cut the appropriate sized band(s) from the gel, place each gel slice in a 1.5 mL tube, and purify the DNA (refer to the Qiagen gel purification protocol; elute with 30 μL EB buffer).
13. Measure the concentration of the purified fragment samples with a Nanodrop Spectrophotometer.
14. Record the absorbance (A260), purity (A260/A280), and concentration (ng/μl) for each sample.

Ligate (paste) the DNA fragments together

1. Calculate how many ng of insert you need to get a 2:1 ratio of insert molecules to 50 ng vector molecules
X ng insert = (bp insert / bp vector) x 2 x 50 ng vector
2. Calculate how many μL of insert and vector you will need for each ligation:
X μL insert = desired ng insert ÷ insert concentration ng/μL (do the same for vector)
3. Set up your ligation reaction(s) in sterile 0.5 mL tubes as shown below:
 Ligation Negative Control Insert DNA (X ng) ___ μL none Vector DNA (50 ng) ___ μL same 2x Roche Rapid Ligation buffer 5.0 μl same NEB T4 ligase 1.0 μl same dH2O ___ μL ___ μL + insert volume 10.0 μL total Mix the reaction(s) thoroughly by flicking the tube.Incubate at room temperature for 10 minutes.

Transform bacteria with the ligated plasmids 30 minutes

1. Warm selection agar plates at 37°C.
2. Incubate DH5α Turbo competent cells on ice just until thawed. Use 30 μL per ligation.
3. Add 30 μL thawed cells to the ligation reaction. Immediately place on ice and incubate for 10 min. (Do not heat shock; No 30 min. recovery is required for Amp resistance)
4. Label the pre-warmed plates with the antibiotic name, strain name, ligation (e.g., "BB part A insert + BB part B vector"), your initials, and the date.
5. Pipette the total volume of cells + ligation onto the agar; speared using sterile glass beads.
6. Incubate overnight at 37°C to get colonies

Note: The negative control will show you the number of “background” colonies so that you can determine whether your transformation worked, or is just the result of vector self-ligation or selection failure.